Dynamic vascular surfaces

Blood vessels have long been considered as passive conduits for blood and circulating cells that, at best, respond to exogenous cytokines. However, recent work has shown that blood vessels serve as a highly dynamic interface between the circulation and tissues. Augustin et al. review molecular mechanisms of vascular development and function in different organs. Differentiated endothelial cells develop as a sort of cobblestone monolayer to form one of the largest surfaces within the body. Vascular control of the tissue microenvironment is vital, not only for normal tissue development and homeostasis, but also for disease states ranging from inflammation to cancer.

Structured Abstract

BACKGROUND

Each organ in the human body has its own capillary bed to carry out its distinctive and versatile functions in response to dynamically changing systemic and local needs. Common and specific functions of the microvasculature in different organs are executed by organ-specifically differentiated endothelial cells (ECs). The morphological differentiation of ECs into barrier-forming continuous ECs, fenestrated ECs, and sinusoidal ECs has long been recognized. Nevertheless, the functional properties and underlying molecular mechanisms of organotypic vasculatures have only been uncovered recently.

ADVANCES

This Review covers recent advances in the biology of organotypically differentiated microvascular beds. It describes the key features of continuous, discontinuous, and sinusoidal ECs, as well as the more specialized ECs of Schlemm’s canal and high endothelial venules. Major transcriptional pathways of EC specification and differentiation are outlined, including GATA4 as a key transcription factor of sinusoidal EC differentiation. The molecular shear stress–sensing machinery—which transduces blood flow–mediated biophysical forces that are essential to maintain the quiescent, differentiated EC phenotype—is delineated.

In terms of function, this Review also discusses discoveries in different organs, including liver, lung, and bone, that have identified organotypically differentiated ECs as a source of paracrine (“angiocrine”)–acting cytokines, through which they exert active gatekeeper roles on their microenvironment. ECs thereby control organ development, homeostasis, and tissue regeneration.

On the basis of these general principles of organotypic vascular differentiation and function, this Review comprehensively covers recent landmark discoveries pertaining to the organotypically specialized (micro)vasculature in different organs. Focusing on the molecular structure-function analysis of organotypically differentiated (micro)vasculatures, it specifically highlights the properties of blood vessels in the brain, eyes, heart, lungs, liver, kidneys, bones, adipose tissue, and endocrine glands. Emphasis is given to the contribution of organotypically differentiated vasculatures to both physiological organ function and disease.

OUTLOOK

Research into the mechanisms of organotypic vascular differentiation and function has emerged in recent years as a new branch of vascular biology, with major implications for our understanding of physiological and pathophysiological organ function. Ongoing research is aimed at deciphering, in much higher resolu­tion (all the way to the single-cell level), the molecular microarchitecture of organotypic vasculatures, understanding the multicellular cross-talk through which organo­typic vasculatures control their microenvironment, dissecting niche functions of organotypic vasculatures with respect to stem cells and their progeny, and unraveling the fate maps of different or­ganotypic vasculatures in health and disease. Future research will not only focus on decipher­ing the molecular mechanisms and functional consequences of organotypic vascular differentia­­tion, but will also aim to translate such knowledge for the develop­ment of novel organ- and vessel bed–specific angiotargeted therapies for multiple diseases that have hitherto been intractable.

Blood vessels in the body (clockwise from top left, vessels in the brain, retina, heart, adrenal gland, bone, and liver) come in different morphologies and have distinct organotypic characteristics that enable them to execute vessel bed–specific functions. They thereby act as gatekeepers of their microenvironments to actively control organ function.

Abstract

Blood vessels form one of the body’s largest surfaces, serving as a critical interface between the circulation and the different organ environments. They thereby exert gatekeeper functions on tissue homeostasis and adaptation to pathologic challenge. Vascular control of the tissue microenvironment is indispensable in development, hemostasis, inflammation, and metabolism, as well as in cancer and metastasis. This multitude of vascular functions is mediated by organ-specifically differentiated endothelial cells (ECs), whose cellular and molecular heterogeneity has long been recognized. Yet distinct organotypic functional attributes and the molecular mechanisms controlling EC differentiation and vascular bed–specific functions have only become known in recent years. Considering the involvement of vascular dysfunction in numerous chronic and life-threatening diseases, a better molecular understanding of organotypic vasculatures may pave the way toward novel angiotargeted treatments to cure hitherto intractable diseases. This Review summarizes recent progress in the understanding of organotypic vascular differentiation and function.

Reflecting the limited diffusion distance of oxygen in tissues, every cell of the body is, with few exceptions (e.g., cartilage), within 100 to 150 μm of the nearest capillary. The vasculature thereby forms a systemically disseminated organ. As a result, essentially all medical disciplines are affected by research in the field of vascular biology. However, vascular biology research is somewhat fragmented and not as coherently developed as would be expected given its importance for human health. In fact, it is still not widely appreciated that dysfunction of the inner lining of blood vessels is the single most common cause of human mortality. The devastating consequences of hypertensive, atherosclerotic, coagulation-related, and pathologic angiogenesis–associated diseases account for more than two-thirds of deaths.

The anatomical heterogeneity of blood vessels in different organs has been recognized for centuries, and research pursued during the past 30 years has, in substantial detail, mapped the molecular repertoire of vessel wall–lining endothelial cells (ECs) in different vascular beds (1, 2). The functional properties and underlying molecular mechanisms of organotypically differentiated ECs have, however, only been uncovered in recent years. It is increasingly recognized that ECs do not just constitute a barrier-forming cell population acting as a responsive interface; rather, they actively control their microenvironment as gatekeepers of organ development, homeostasis, and tissue regeneration (3). This paradigm shift will guide future research aimed at exploiting the organotypic vasculature as a therapeutic target for a broad spectrum of vascular and organ diseases. This Review summarizes the latest findings and advances in the mechanistic understanding of organotypic vascular properties and function, highlighting key features of the vasculature in different organs in development, maintenance, homeostasis, and disease. It is not aimed at comprehensively covering the vasculature of all organs, but it instead focuses on organotypic vasculatures for which there have been major and groundbreaking discoveries in recent years. We mostly focus on ECs, which should not detract from the fact that surrounding pericytes are increasingly recognized as major contributors to organotypic vascular structure and function (4). This Review is restricted to the blood vascular system and does not cover recent advances in the field of lymphatic biology, including the landmark discovery of a lymphatic system in the brain (5, 6), which are reviewed elsewhere (7, 8).

Characteristics of organotypic capillaries

Capillaries have a diameter of 5 to 10 μm and are lined by a single layer of ECs. There are three major types of capillaries: continuous, fenestrated, and sinusoidal (Fig. 1). Barrier-forming continuous capillaries exist ubiquitously in the human body, except in epithelia and cartilage (Fig. 1A). The distinctive architecture of these capillaries permits the diffusion of water, small solutes, and lipid-soluble materials into the surrounding tissues and interstitial fluid without any loss of circulating cells and plasma proteins. Larger molecules such as glucose and other nutrients pass through the EC monolayer by transcytosis, a process regulated by specific transporters. Continuous capillaries exist in a more specialized state in most of the central nervous system; the ECs are firmly bound together by tight junctions because of the necessity of stricter permeability that precludes most large molecules, drugs, and pathogens from passing.

Endothelial cells (ECs) are shown in light pink, encircled in black. (A) Barrier-forming continuous capillaries are found in most organs, including the brain and retina. Solutes are transported through the EC monolayer by regulated transcytosis. (B) Fenestrated capillaries have intracellular pores that are covered with a diaphragm; this structure is found in the endocrine glands, intestine, and kidneys, rendering them permeable by fluid and small molecules. (C) Sinusoidal capillaries have larger intercellular gaps and a discontinuous basement membrane, allowing free exchange of materials; this structure is generally found in the liver, bone marrow, and spleen. (D) Schlemm’s canal enables sufficient aqueous humor outflow from the anterior eye chamber by means of high-rate transcytosis with numerous giant vacuoles. (E) High endothelial venules (HEVs), lined with cuboidal ECs, serve as an entry point for lymphocyte migration from the blood into lymph nodes. Some lymphocytes accumulate transiently in “HEV pockets.” The orange dashed arrows indicate the main directions of transcytosis, flow, or lymphocyte migration.

Fenestrated capillaries have intracellular pores (“windows”) with a diaphragm that penetrate the endothelial lining (Fig. 1B). The pores not only speed up the exchange of water, but also permit the passage of solutes as sizable as small peptides between plasma and interstitial fluid. This structure is observed with varying permeability and numbers of pores in the choroid plexus of the brain; several endocrine organs such as the pineal, pituitary, and thyroid glands; the hypothalamus; filtration sites in the kidneys; and absorptive areas of the intestinal tract.

Although closely resembling fenestrated capillaries, sinusoidal endothelium has gaps instead of pores between ECs and is characterized by flattened and irregular shapes and inadequate coverage by thinner basal lamina (Fig. 1C). Such characteristics lead to free exchange of water and provide a conduit for large solutes such as plasma proteins between plasma and interstitial fluid. Sinusoids are located in the liver, spleen, bone marrow, and several endocrine organs, including the pituitary gland and the adrenal medulla. Because of the requirement of extensive exchange in these organs, the blood current decelerates in sinusoids to extend the time of exchange across the sinusoidal barrier. In the meantime, phagocytic cells that are distributed along the sinusoids of the liver, spleen, and bone marrow act as wardens that detect and engulf exogenous pathogens, damaged cells, and debris. In comparison, some exchange may occur between blood and interstitial fluid or aqueous fluid by bulk transport—the transcellular movement of vesicles that form through endocytosis (transcytosis) at the inner endothelial surface.

The transcriptional machinery of early embryonic differentiation is overall relatively well understood, but much needs to be learned about the interplay between developmental vascular transcription factors. Similarly, little is known about the transcriptional programs driving organ-specific EC specification and differentiation. Recently, the transcription factor GATA4 was identified as a master regulator of hepatic microvascular specification and acquisition of organ-specific vascular competence (29). GATA4 mediates the down-regulation of continuous EC-associated transcripts and up-regulates sinusoidal EC genes. This landmark discovery was only made possible through the generation and use of Stab2-Cre mice for the conditional deletion of GATA4 selectively in sinusoidal ECs (29). The functional analysis of molecules controlling organotypic EC differentiation is hampered by the lack of Cre driver mice for specific subpopulations of ECs.

Flow-dependent maintenance of the organotypic vasculature

Blood flow–mediated biophysical forces, erroneously called shear “stress,” are essential to maintain the quiescent, differentiated EC phenotype (30, 31). Shear stress–induced nitric oxide (NO) production and release was identified more than 20 years ago as a regulator of vascular tone and thereby a contributor to vessel maintenance (32, 33). More recently, hemodynamic forces have been shown to control hematopoiesis in a NO-dependent manner (34, 35).

Molecular analysis of EC gatekeeper functions in the microenvironment is still in the early stages, and much needs to be learned. For example, what is the role of aberrant production of angiokines by locally diseased ECs in the control of organ function? How do differences in young and aged ECs contribute to changes in angiocrine signaling during aging? How are epigenetic mechanisms such as epigenetic memory involved in the regulation of EC angiocrine functions? Recent advances in omics techniques allowing the profiling of single-cell transcriptomes (49) make it possible to address these questions, which will generate insights into the mechanisms and functional consequences of organotypic EC differentiation at a hitherto unparalleled level of resolution.

Blood-brain barrier–forming vasculature

Supported by pericytes and astrocytes, the brain endothelium forms a particularly tight layer called the blood-brain barrier (BBB) (50, 51). ECs of the BBB have specialized tight intercellular junctions, no fenestrae, and an extremely low rate of transcytosis. These special features protect the brain from harmful substances but are also an obstacle for the delivery of therapeutic drugs into the brain. BBB ECs express specialized molecular transporters and receptors such as GLUT-1 (glucose transporter type 1) and members of the ABC (adenosine triphosphate–binding cassette) transporter family. For example, docosahexaenoic acid (DHA) is an omega-3 fatty acid essential for normal brain growth and cognitive function. It cannot be de novo synthesized in the brain and must be transported across the BBB. Although the mechanisms for DHA uptake into the brain had been enigmatic until recently, MFSD2A has been identified as the major transporter (52), as well as a negative regulator of transcytosis (53).

The formation and maintenance of the BBB require several specific molecules, including GPR124 (G protein–coupled receptor 124) (54, 55). GPR124 functions as a coactivator of Wnt7a- and Wnt7b-stimulated canonical Wnt signaling via a Frizzled receptor and LRP co-receptor acting in concert with Norrin/Frizzled4 signaling to control vascular development of the central nervous system and BBB maintenance (56). Ablation of Norrin/Frizzled4 signaling results in BBB defects, which are ameliorated by stabilizing Β-catenin (57).

BBB breakdown is a hallmark of several neurological disorders including ischemic stroke, multiple sclerosis, and Alzheimer’s disease (58–60). Recent advances in the pathogenesis of cerebral cavernous malformations (CCMs) have provided important insights into BBB maintenance and breakdown mechanisms. CCMs have a prevalence of up to 0.5% in the general population. Loss of function in any of the three genes KRIT1 (CCM1), CCM2, and PDCD10 (CCM3) causes familial CCMs. Mechanistically, mutations in the three nonhomologous genes converge in the gain of MEKK3 (MAPK/ERK kinase kinase 3)–KLF2/4 signaling in early CCM lesions, perturbing the quiescent EC phenotype (61). EC quiescence during CCM progression is also controlled by the vascular destabilizing functions of ANGPT2, whose UNC13B- and VAMP3 (vesicle-associated membrane protein 3)–dependent exocytosis is actively suppressed by PDCD10 (62). Vascular destabilization induced by EC-specific deletion of KRIT1 (CCM1) promotes endothelial-to-mesenchymal transition through up-regulation of BMP6, TGFΒ, and BMP signaling (63). Endothelial-to-mesenchymal transition is a result of KLF4 transcriptional activity, which promotes TGFΒ/BMP signaling through the production of BMP6 (64). Recently, EC TLR4 (Toll-like receptor 4) and the gut microbiome were identified as critical upstream stimulants of CCM formation (65).

Blood vessels in the eye

Dysfunction and concomitant angiogenesis of the retinal vasculature is a major cause of blinding diseases, including diabetic retinopathy and age-related macular degeneration (AMD). In mice, retinal vessels grow postnatally in a highly organized pattern, allowing the study of physiological developmental angiogenesis in a quasi–two-dimensional setting. This model has been widely used to decipher the molecular and cellular mechanisms of sprouting angiogenesis, tip and stalk cell formation, artery versus vein specification, and vascular remodeling and pruning. Retinal vessels exist as superficial, intermediate, and deep vascular plexuses (Fig. 2A). Upon formation of the superficial plexus, vertical sprouting into the deep layers initiates, sequentially forming intermediate and deep vascular plexuses. The hypoxia-driven VEGF gradient acts as an angiogenic cue that stimulates tip cells to extend, along with preformed astrocytes, during postnatal development. Premature babies who receive oxygen therapy often develop retinopathy of prematurity as a result of disorganized vessel growth such as vasoregression and abnormal angiogenesis (Fig. 2A). EC-derived Wnt ligands and FGD5 are closely involved in vascular pruning and regression (22, 66) (Fig. 2A). In contrast, integrin αvΒ8 expressed in astrocytes and SLIT2 secreted from surrounding neuronal cells stimulate and stabilize the three retinal vascular plexuses (67–69). Surrounded by pericytes, glial cells, and neurons, the retinal vessels form the blood-retinal barrier (BRB), which prevents the diffusion of toxic compounds and microorganisms and proinflammatory leukocytes from surrounding tissues. Patients with diabetic retinopathy exhibit multiple hallmarks of vasculopathies such as pericyte dropout, microaneurysm, nonperfused vessels, and abnormal angiogenesis in the retina (Fig. 2A). Inadequate pericyte coverage impairs retinal vascular growth and BRB function, leading to ANGPT2 up-regulation and sensitization to VEGF, which triggers the progression of BRB breakdown (70).

The choroid has the largest blood flow by weight of all organs and an extremely anastomosed capillary network called the choriocapillaris. ECs of the choriocapillaris have numerous fenestrations that permit the passage of large molecules into the extravascular space. The distribution of choroidal pericytes is extremely diverse, ranging from complete coverage in primary arteries to sparse dispersion in the choriocapillaris (71). Although this distribution may contribute to the structural plasticity of the choriocapillaris in response to physiological changes, sparse pericyte ensheathment could be related to neovascular (NV-) AMD. NV-AMD is characterized by choroidal neovascularization, an outgrowth of leaky vessels from the choriocapillaris, penetrating through Bruch’s membrane, which gradually disintegrates during AMD progression; this leads to serous or hemorrhagic leakage, retinal edema, and subsequent visual dysfunction (Fig. 2B). In NV-AMD, loss of the choriocapillaris causes ischemia in retinal pigment epithelium (RPE), followed by the production of hypoxia-inducible angiogenic factors, including VEGF, in RPE and adjacent cells (72). Consequently, VEGF blockade is the treatment of choice at present for NV-AMD. VEGF is, however, also required to maintain the choroidal vasculature, and repeated treatment with VEGF blockers could aggravate hypoxia and oxidative stress by inducing damage of the choriocapillaris, leading to vision loss caused by dysfunction of photoreceptors (73). The number of patients with NV-AMD is rapidly increasing. Thus, safer and more effective treatments are needed.

Corneal avascularity is essential for the cornea’s transparency and unperturbed vision. Avascularity is presumably due to an environment rich in antiangiogenic and antilymphangiogenic molecules, including truncated soluble VEGFR1, VEGFR2, and VEGFR3 secreted from surrounding cells (74, 75). Corneal neovascularization, the excessive ingrowth of blood and lymphatic vessels from the limbus, occurs in several pathologic conditions, such as herpes simplex stromal keratitis, contact lense–induced keratitis, and graft rejection after keratoplasty (76) (Fig. 2C). Although anti-VEGF therapy has shown promising effects in patients with corneal neovascularization, further investigation is needed to limit corneal scarring and loss of transparency.

Schlemm’s canal is an endothelium-lined channel that encircles the cornea and provides a specialized vascular bed for aqueous humor flow, which constantly refreshes the anterior eye chamber between the cornea and the lens (Figs. 1D and 2D). Aqueous humor is produced by the ciliary body and is drained through a trabecular meshwork into Schlemm’s canal and aqueous and episcleral veins. Schlemm’s canal shares morphological and functional attributes of lymphatic vessels (77–79). Molecularly, Schlemm’s canal is an intermediate vessel type between lymphatic and blood vessels, expressing PROX1, VEGFR3, and integrin α9, but not LYVE-1 or podoplanin (77–79). The primitive Schlemm’s canal originates from the choroidal vein, and Schlemm’s canal endothelium is postnatally respecified to acquire lymphatic traits by up-regulating PROX1 (79). Schlemm’s canal dysfunction, including reduced giant vacuole formation, results in impaired aqueous humor drainage and increased intraocular pressure, ultimately leading to glaucoma (Fig. 2D). Intriguingly, primary congenital glaucoma is also detected in mice with genetic deletion of Angpt1 and Angpt2 or deletion of the angiopoietin receptor gene Tie2 (80). Tie2 mutations have been identified in patients with primary congenital glaucoma (81), potentially paving the way for novel therapeutic strategies for glaucoma.

Heart vasculature

The origin of cardiac capillary ECs has been the subject of intense study in recent years. The proepicardium was proposed as the source of cardiac capillaries, but this has been challenged by recent lineage-tracing analyses in mice. Only a subset of the proepicardium contributes to a small portion of cardiac capillaries (82–84), whereas the sinus venosus and the endocardium serve as primary sources. The sinus venosus provides EC progenitors for cardiac capillaries, which further contribute to a substantial portion of the cardiac capillary bed in the lateral free walls of the ventricles (85–87) (Fig. 3, A and C). Endocardial progenitors give rise to cardiac capillaries within the ventricular septum and ventral wall of the embryonic heart (87, 88) (Fig. 3B). Interestingly, endocardial cells are converted to ECs of newly formed cardiac capillaries in the inner ventricular wall during trabecular compaction in the neonatal heart (89).

(A) Migrating ECs from the sinus venosus form a capillary bed over the dorsal surface of the growing heart and infiltrate into the myocardium of the ventricular wall (C). (B) Endocardial cells contribute to the establishment of most coronary vessels in the ventricular septum. (C) During capillary invasion into the myocardium, a subset of the capillary bed develops into coronary arteries, which are covered by pericytes that differentiate into smooth muscle cells (SMCs). The majority of pericytes are derived from epicardial cells.

The molecular mechanisms of cardiac vascular morphogenesis remain poorly understood. Myocardium-derived VEGF regulates the endocardial contribution to the origin of cardiac capillaries (88). Epicardial VEGF-C promotes sprouting angiogenesis from the sinus venosus (87). Moreover, myocardium-derived ANGPT1 controls coronary vein formation (90). Mural cells [pericytes and smooth muscle cells (SMCs)] provide structural support to adjacent endothelium and maintain vessel lumen. Unlike cardiac capillary ECs, the majority of cardiac mural cells are derived from epicardial cells (82, 83). Lineage tracing has also revealed that cardiac pericytes develop into SMCs of coronary arteries in response to Notch signaling during arterial remodeling; this study also showed the presence of undifferentiated pericytes in the adult heart, which can be used to produce SMCs during the regeneration of coronary arteries (91).

Coronary atherosclerosis causes myocardial infarction and subsequent heart failure, one of the leading causes of death worldwide. Atherosclerotic plaques with neointima formation are the most prominent pathologic substrate. Yet growing evidence suggests that ECs play a pivotal role in the pathogenesis of atherosclerosis (92, 93). Laminar shear stress maintains endothelial quiescence and suppresses atherosclerosis through activation of KLF2, which induces endothelial NO synthase (eNOS) and thrombomodulin (94). KLF2 coordinates atheroprotective communication between ECs and SMCs by regulating microRNA-143 (miR-143) and mi-145 expression, thereby controlling SMCs (95). Endothelial miR-126 limits atherosclerosis by targeting the noncanonical Notch ligand DLL1 (96). Unidirectional laminar shear stress also induces phosphorylation and suppression of YAP/TAZ through integrin signaling (41). Disturbed flow accelerates the progression of atherosclerosis by transcriptional activation of YAP/TAZ signaling and proinflammatory gene expressions, which can be reversed by YAP/TAZ inhibition (41).

Blood-gas barrier in the lungs

The circulatory system of the lungs is composed of a thin layer of capillary ECs underlying an expansive surface of alveolar epithelial cells. The development of the lungs requires precise temporal and spatial organization of capillary ECs and alveolar epithelial cells. Proper capillary density and positioning is specified early in development and is required for viability at birth (100). Angiocrine factors secreted from primitive lung capillaries contribute to specifying endoderm and mesoderm progenitor differentiation into primitive lung epithelial and vascular precursor cells (101, 102). Likewise, BMP4-BMPR1A signaling triggers calcineurin/NFATc1–dependent expression of TSP1 (thrombospondin 1) in lung ECs to drive alveolar lineage–specific differentiation of bronchoalveolar stem cells (103). During postnatal development of the lung, a high level of vascular refinement, remodeling, and maturation is imperative to determine the tissue-specific architecture of branched organs, which is independent of perfusion, flow, or blood-borne substances (104).

Defective lung vascular development can result in alveolar capillary dysplasia (ACD) and bronchopulmonary dysplasia (BPD). ACD is a lethal disorder in humans characterized by a failure of alveolar capillary formation, often accompanied by misalignment of pulmonary veins. BPD is a chronic lung disease affecting premature infants (<1000 g) that is caused by impaired alveolarization and dysmorphic vascular development associated with prematurity (105). The development of therapeutic strategies to repair the respiratory capacity in patients with lung disorders is handicapped by the limited understanding of lung regeneration mechanisms. Reconstitution of the alveolar-capillary interface is pivotal for adult lung regeneration (45). Transplantation of properly activated lung capillary ECs or injection of lung-specific angiocrine mediators could therefore emerge as a therapeutic modality to drive epithelial cell repopulation and improve respiratory function (3, 45).

Liver vasculature

Endowed with two separate, albeit connected, vascular systems—the arterial and the portal vasculature—the liver has a distinctive vascular supply. The arterial system primarily serves nutritive purposes, whereas the portal system feeds lipid droplet–rich, poorly oxygenated blood from the intestine to the liver. Both systems drain blood through the sinusoidal vasculature (Figs. 1C and 4). The sinusoidal vasculature has a characteristic morphology, with fenestrations grouped into sieve plates, specialized junctional complexes, and an incomplete basement membrane. Sinusoidal fenestrae allow free flow of blood (except cells) into the space of Disse, with direct access to the surface of hepatocytes. They also enable T lymphocytes to send foot processes into the space of Disse, allowing immunosurveillance of hepatocytes without extravasation (106).

Fig. 4Positional relationship between blood vessels and hepatocytes in the liver.

Lipid droplet–rich blood from the portal vein (PV) mixes with blood from the hepatic artery (HA) and flows through the sinusoidal vasculature into the central vein (CV). Fenestrated liver sinusoidal ECs (LSECs; inset) are separated by the space of Disse from the hepatocytes, which are zonated along the sinusoidal vasculature. Liver zonation is controlled by EC-derived signals involving Wnt ligands and the Wnt signaling enhancer RSPO3. Liver regeneration is controlled by angiocrine signals secreted from ECs. BD, bile duct; Hep, hepatocyte; KC, Kupffer cell; HepSC, hepatic stellate cell.

Liver sinusoidal ECs (LSECs) make up a heterogeneous cell population (107) that serves important scavenger functions, clearing macromolecular waste molecules from the circulation. LSECs also act as antigen-presenting cells and mediate clearance of immune complexes, viruses, lipopolysaccharides, and other molecules (108). These functions are executed by different scavenger receptors [e.g., STAB1 and STAB2 (stabilin 1 and 2)] and other molecules mediating highly efficient endocytosis (109). Perturbation of scavenger receptor function (e.g., by genetic deletion of STAB1 and STAB2) has systemic consequences and causes glomerulofibrotic nephropathy (110).

The liver vasculature has been extensively studied for its ability to control organ function in an angiocrine manner. During embryonic development, paracrine signals from ECs control early steps of organogenesis (111). The transcription factor GATA4 drives LSEC specification, which is an indispensable requirement not just for normal liver development, but also for liver colonization by hematopoietic progenitor cells. Mice with targeted deletion of GATA4 in LSECs die from anemia in late embryonic development (29). Likewise, LSEC-expressed PLVAP (plasmalemma vesicle–associated protein) is essential for the seeding of fetal monocyte-derived macrophages to tissues in mice. PLVAP forms diaphragms in the fenestrae of liver sinusoidal endothelium during embryogenesis and selectively regulates the egress of fetal liver monocytes to the systemic vasculature (112).

Lineage-tracing experiments in adult mice have identified AXIN2-positive pericentral hepatocytes as a self-renewing reservoir of cells that replace hepatocytes during homeostatic renewal. Proliferation of these cells is controlled by Wnt ligands produced by central vein ECs (113). Central vein ECs are also the primary source of the Wnt signaling enhancer RSPO3, thereby controlling liver zonation (114). It remains to be seen how morphogenetic Wnt signaling gradients originate from the central vein against the primary direction of blood flow, but the retrograde flow in the space of Disse is likely involved in this process (Fig. 4). As a result, hepatocytes receive their characteristic zonal alignment along the sinusoids, which has been mapped at single-cell resolution to reveal that around 50% of liver genes are nonuniformly expressed, in line with this zonation (115). Future complementary single-LSEC mapping will enable the construction of a bioinformatic signaling cross-talk map to shed new light on the molecular mechanisms of stromal-parenchymal interaction. For example, BMP2 secreted by LSECs acts on hepatocytes to control local and systemic iron metabolism (116). This example likely reflects only the tip of the iceberg, and it can be expected that numerous other EC-derived instructive gatekeeper functions will be identified in the future.

Beyond homeostatic maintenance, LSECs regulate the response to liver damage (117). Partial hepatectomy leads to rapid global proliferation of the remaining hepatocytes. Angiocrine signals including HGF and Wnt2, secreted from LSECs before the induction of angiogenesis, stimulate hepatocyte proliferation (118, 119). Similarly, down-regulation of ANGPT2 in LSECs after partial hepatectomy leads to down-regulation of LSEC TGFß production (43). TGFß is a potent negative regulator of hepatocyte proliferation. As such, LSEC down-regulation of TGFß enables liver regeneration by removing an angiocrine break. Intriguingly, recovery of LSEC ANGPT2 production during the later steps of liver regeneration controls LSEC VEGFR2 expression in an autocrine manner, thereby facilitating the angiogenic response to hepatocyte-derived VEGF (43). As such, the dynamic temporal regulation of a single LSEC-derived cytokine controls hepatocyte and LSEC proliferation as a dynamic rheostat at different stages of liver regeneration.

Long-term liver damage results in fibrosis, which is a major risk factor for the development of hepatocellular carcinoma (HCC). Fibrosis and HCC lead to LSEC transdifferentiation with loss of LSEC markers and sinusoidal fenestrae, a process known as capillarization, during which LSECs lose their protective properties and promote angiogenesis and vasoconstriction (120, 121). As a result, LSEC dedifferentiation is associated with a switch from vessel co-option and intussusceptive angiogenesis to sprouting angiogenesis (122, 123). LSEC-derived angiocrine signals contribute to regulating the balance between liver regeneration and fibrosis (42). The regulatory loops between the vascular and parenchymal compartments are, however, not restricted to LSEC-hepatocyte interactions, but also involve hepatic stellate cells (HepSCs), which are the specialized pericytes of the liver. For example, HepSC-expressed endosialin promotes fibrogenesis and reduces hepatocyte proliferation during liver damage (124). Conversely, activated HepSCs limit HCC progression in an endosialin-dependent manner (125).

Kidney microvasculature

The renal vasculature supports kidney function in distinctly different compartments, including glomeruli, cortical peritubular capillaries, and vasa recta bundles. Correspondingly, different types of capillaries are found in different parts of the kidney. Glomerular ECs are the prototype of fenestrated endothelium. For efficient filtration of serum to Bowman’s space, glomerular capillaries form compact loops to maximize the contact surface of blood flow to the glomerular filtration unit. The basement membrane of glomerular ECs is fused to that of podocytes to facilitate filtration, whereas the outer surface of glomerular ECs is wrapped by foot processes of podocytes to form an additional barrier of filtration. VEGF/VEGFR2 signaling is essential for embryonic development and maintenance glomeruli. VEGF blockade may cause proteinuria and thrombotic microangiopathy (126). Increased levels of VEGF have been detected in diabetic nephropathy, although whether VEGF actually contributes to disease progression remains inconclusive (127). Moreover, VEGF secreted from proximal tubules contributes to the maintenance of peritubular capillaries and blood pressure (128). Soluble extracellular domain of VEGFR1 (sFlt1) is produced by glomerular podocytes and dynamically regulates local VEGF by acting as a decoy receptor of VEGF (129). Inhibition of podocyte-secreted sFlt1 causes disintegration of the glomerular barrier, leading to proteinuria and renal failure such as nephrotic syndrome as a result of cytoskeleton reorganization in podocytes (129). In addition to VEGF/VEGFR2 signaling, ANGPT1/TIE2 signaling is critical for glomerular development and maintenance. Genetic inactivation of ANGPT1 results in dilated glomerular capillary loops (130). ANGPT1 secreted by podocytes and mesangial cells acts protectively against diabetic kidney injury (131).

Bone vasculature

Long bones are highly vascularized, except in the growth plate and articular cartilage. The vasculature has a hierarchical organization, with arterial feeding into a capillary network and drainage into a large central vein in the diaphysis. Nutrient arteries penetrate the medullary cavity through the bone cortex and run toward the metaphysis as interconnected columnar capillaries. Columnar capillaries make an arch near the growth plate, forming a sinusoidal network that drains into the central vein. Endochondral ossification is an example of angiogenic-osteogenic coupling at the primary ossification center (embryonic day 14 in mice), growth plate, and secondary ossification center (postnatal day 6 in mice) (Fig. 5A).

Adult bone capillaries can be divided into two types, H and L, on the basis of their marker expression and function (136) (Fig. 5B). ECs in type H vessels express high levels of CD31 and endomucin. Type H vessels are primarily in the metaphysis and endosteum, where they are closely associated with PDGFRß (platelet-derived growth factor receptor ß)–expressing perivascular cells and osterix-expressing osteoprogenitor cells. In contrast, ECs of type L vessels express lower levels of CD31 and endomucin. Type L vessels are highly branched and form the sinusoidal vascular network in the diaphysis, where they are surrounded by hematopoietic cells (139). Because arteries exclusively supply type H vessels, type L vessels receive blood from type H vessels; thus, each type of vessel has a distinct metabolic environment. Type H vessels in the metaphysis and endosteum are relatively well oxygenated, whereas type L vessels in the diaphysis are in a low-oxygen environment because of the lack of a direct arterial supply (44, 136). The blood velocity is also higher in type H vessels, suddenly dropping after the branching point in type L sinusoidal vessels (140). Variations in oxygen tension and permeability create distinctly different microenvironments for hematopoietic stem cells (HSCs) in the bone marrow. Less permeable arterial blood vessels maintain HSCs in a state with low reactive oxygen species, whereas the more permeable sinusoids promote HSC activation and serve as sites for immature and mature leukocyte trafficking to and from the bone marrow (141).

Bone undergoes constant remodeling that is regulated by the balanced activity of osteoblasts (bone formation) and osteoclasts (bone resorption). Disruption of this balance may cause age-related and disease-associated bone losses that increase the risk of fracture (142). Bone resorption increases with aging, primarily as a result of hormone changes, whereas bone formation gradually decreases. In addition, type H vessels and perivascular osteoprogenitors are reduced in aged animals without a substantial reduction in the total number of ECs (136). Decreased blood flow in aged mice may be associated with decreased bone formation and loss of type H vessels (Fig. 5B). Furthermore, type H vessels are reduced in a premenopausal mouse model of osteoporosis (143). Aging results in the strong reduction of HSC niche–forming vessels. EC Notch signaling supports the expansion of HSC niches, and activation of EC Notch signaling restores HSC-supporting vascular niches (44, 144). Thus, pharmacological rejuvenation of blood vessels may become an attractive therapeutic goal to prevent age-related or disease-related loss of bone mass.

Blood vessels in adipose tissue

Adipose tissue is one of the most plastic organs, constantly expanding and shrinking depending on energy status. Adipogenesis is highly dependent on angiogenesis (145, 146) (Fig. 6A). The vasculature of adipose tissue provides external cues for the development and differentiation of adipose progenitors as a vascular niche (147, 148). In turn, adipocytes produce angiogenic factors during adipose tissue expansion and remodeling (149). Adipocyte-derived VEGF and high VEGFR2–expressing adipose vasculatures play crucial roles in adipogenesis, lipogenesis, acclimation to cold exposure, and metabolic functions (150). Decreased VEGF-B signaling in rodent models of type 2 diabetes has been reported to restore insulin sensitivity and improve glucose tolerance (151). More recent work has challenged these findings, suggesting that VEGF-B gene transduction inhibits obesity-associated inflammation and improves metabolic health without changes in body weight or ectopic lipid deposition (152).

(A and B) Angiogenesis governs adipogenesis to differentiate preadipocytes into mature adipocytes. These mature adipocytes can develop into either hypertrophic adipocytes as a result of a high-fat diet–induced increase in lipid accumulation or into beige adipocytes upon cold exposure or Β3-adrenergic receptor agonism. These processes are accompanied by vascular changes; a high-fat diet decreases vascular density, whereas browning by cold exposure increases vascular density in adipose tissues. This plastic transition of adipocytes from brown or beige adipocytes to white adipocytes and vice versa is termed whitening and browning, respectively. (C) Expression of vascular fatty acid transporters (FATPs) induces subsequent transport of FAs across the EC layer into cells of metabolic organs. GPIHBP1 shuttles lipoprotein lipase (LPL) from interstitial spaces to the bloodstream, which subsequently hydrolyzes triglycerides (TGs) into FAs, possibly mediated by FATP3 and FATP4 during transendothelial FA transport.

The vasculature in adipose tissue serves as a rich source for paracrine-acting hormones, growth factors, and inflammation-related cytokines. In obesity, expanded white adipose tissue (WAT) displays capillary rarefaction and hypoxia, which correlate with inflammatory cytokine expression and macrophage infiltration (150) (Fig. 6A). In contrast, hypervascularization of adipose tissue can be associated with browning (149) (Fig. 6A). Brown adipose tissue (BAT) is a highly vascularized organ with abundant mitochondria that produce heat through UCP1 (highly enriched uncoupling protein 1)–mediated uncoupled respiration. An increase in lipid accumulation caused by a high-fat diet or the absence of heat stress (thermoneutrality) reduces the rate of thermogenesis, leads to deposition of excess calories as lipids, and causes whitening, which is the transition from BAT to WAT characteristics (153) (Fig. 6A).

The adipose endothelium is a gatekeeper between blood and adipose tissue. EC-regulated fatty acid (FA) transport and uptake limits excessive lipid accumulation, lipid overflow in the bloodstream, and, consequently, insulin resistance (154). FA transporters (FATPs) mediate transendothelial FA transport, and FATP3 is the only FATP that is expressed specifically in blood vessels (155) (Fig. 6B). Moreover, EC-expressed GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1) is crucial for the lipolytic processing of triglyceride-rich lipoproteins (156) (Fig. 6B). Thus, endothelial metabolic functions and the mechanisms involved in the cross-talk between ECs and adipocytes could have important implications for the treatment of obesity, diabetes, and metabolic syndrome.

Endocrine glands

Endocrine glands are endowed with a dense network of capillaries, which provide substrates for hormone synthesis and transport released hormones (157). Their fenestrated capillaries facilitate proper transport of low-molecular-weight hydrophilic molecules, including synthesized hormones. ECs and pericytes in endocrine glands secrete a battery of growth factors, cytokines, and ECM molecules, which control proliferation, differentiation, maintenance, and even regeneration of endocrine cells. Maintenance of endocrine vascular integrity is tightly regulated by VEGF/VEGFR2 signaling (158). Stimulating hormones such as TSH, ACTH, and FSH increase transcellular transport by increasing EC content and the number of intact fenestrae on each EC through up-regulation of VEGF in glandular epithelial cells of the thyroid, adrenal glands, and ovaries (luteinizing granulosa cells). Conversely, low levels of stimulating hormones decrease transcellular transport by reducing EC content and the number of fenestrae on each EC as a result of reduced expression of VEGF in glandular epithelial cells. However, the molecular mechanisms of fenestrae formation and maintenance have not been unraveled in great detail. Long-term inhibition of VEGF or VEGFR2 eventually leads to the reduction of EC fenestrae, resulting in hypofunction of endocrine glands (158–160). The safe margin of VEGF or VEGFR2 blockade—for example, in the treatment of cancer patients—for proper endocrine function and maintenance of EC fenestrae needs to be better defined.

Conclusion and perspective

Blood pressure regulation, coagulation, inflammation, atherosclerosis, and angiogenesis are the five major branches of vascular biology research. The recent advances in the understanding of the molecular mechanisms driving organotypic vascular differentiation and function reflect the emergence of a sixth branch. Organotypic vascular research will be aimed at (i) unraveling, in much greater mechanistic detail, the molecular repertoire of organotypically differentiated cells of the vessel wall (not restricted just to ECs) all the way to the single-cell level; (ii) elucidating the multidirectional molecular cross-talk of vessel wall cells with the cells of their microenvironment, as well as systemic effects controlled by organotypic vasculatures; (iii) dissecting niche functions of organotypic vasculatures on stem cells and their progeny; and (iv) resolving the fate maps of different organotypic vasculatures in health and disease. It will thereby lay the foundation for novel diagnostic and capillary bed–specific angiotargeted therapies.

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Schlemm’s canal is a unique vessel with a combination of blood vascular and lymphatic phenotypes that forms by a novel developmental process. PLOS Biol.12,
e1001912 (2014). 10.1371/journal.pbio.1001912pmid:25051267

Acknowledgments: We regret that, because of space limitations, we were not able to cite all of the original research articles and related references on this topic. Work in the authors’ laboratories is supported by funds from the Deutsche Forschungsgemeinschaft, the Helmholtz Association, the European Union, the Fondation Leducq, and the Institute for Basic Science funded by the Ministry of Science, ICT and Future Planning, Korea (grant IBS-R025-D1).